S. Typhimurium Research Articles

Salmonella Gallinarum mgtC mutant shows a delayed fowl typhoid progression in chicken

Salmonella Gallinarum (SG) provokes fowl typhoid, an infectious disease of acute clinical course that affects gallinaceous of any age and leads to high mortality rates. During the typhoid-like systemic infection of S. Typhimurium (STM) in mice, the bacterium expresses the mgtC gene, which is encoded in the Salmonella Pathogenecity Island – 3 (SPI-3). In this serovar, the function is linked to bacterial replication within macrophages, and its absence attenuates the pathogen. We hypothesized that deleting mgtC from SG genome would alter the microorganism pathogenicity in susceptible commercial poultry in a similar manner. Thus, the present study sought to elucidate the importance of mgtC on SG pathogenicity. For this, a mgtC-mutant lacking S. Gallinarum mutant was constructed (SG ΔmgtC). Its ability to replicate in medium that mimicries the mgtC-related intracellular environment of macrophages as well as in primary macrophages from chicken was evaluated. Moreover, the infection of susceptible chickens was performed to elucidate its pathogenicity and the elicited immune responses by measuring key interleukins by qRT-PCR and the population of macrophages and lymphocytes T CD4+ and CD8+ by means of immunohistochemistry. It was observed that mgtC was required for S. Gallinarum replication in acidified low-Mg2+ media and survival within macrophages. However, unlike its requirement for initial phase of STM infection in mice, lower bacterial counts were only observed at the late stage of macrophage infection without affecting the citotoxicity. Experiments showed that knocking-out the mgtC gene neither altered bacterial uptake by macrophages nor affects bacterial counts in liver and spleen and total chicken mortality. However, plotting a survival curve and analyzing the clinical-pathologic conditions, it was observed a slower progression of the disease in chickens infected by SG ΔmgtC compared to those challenged by the wild-type strain. Furthermore, the mRNA expression of IFN-γ and LITAF were similar between the infected chickens, but higher than in the uninfected group. The same was observed in macrophages and lymphocytes T CD4+ populations. On the other hand, the presence of lymphocytes T CD8+ was increased in the initial phase of the disease provoked by the wild-type strain over the mutant strain. We concluded that the role of mgtC in Fowl Typhoid in susceptible chickens differs from the role in typhoid-like infections in mammals. Thus, the deletion of mgtC gene from S. Gallinarum genome does not affect the overall pathogenicity, but slightly alters the pathogenesis.

Isolation, characterization, and application of a novel, lytic phage vB_SalA_KFSST3 with depolymerase for the control of Salmonella and its biofilm on cantaloupe under cold temperature

This study investigated the efficacy of a novel Salmonella phage with depolymerase activity to control S. Typhimurium (ST) and its biofilm on cantaloupes, for the first time, under simulated cold temperature. vB_SalA_KFSST3 forming a halo zone was isolated and purified from a slaughterhouse with a final concentration of 12.1 ± 0.1 log PFU/mL. Based on the morphological and bioinformatics analyses, vB_SalA_KFSST3 was identified as a novel phage belonging to the family Ackermannviridae. Before employing the phage on cantaloupe, its genetic characteristics, specificity, stability, and bactericidal effect were investigated. Genetic analyses confirmed its safety and identified endolysin and two depolymerase domains possessing antibiofilm potential. In addition, the phage exhibited a broad specificity with great efficiencies toward five Salmonella strains at 4 °C, 22 °C, and 37 °C, as well as stable lytic activity over a wide range of pHs (3 to 11) and temperatures (−20 °C to 60 °C). The optimal multiplicity of infection (MOI) and exposure time of phage were determined to be 100 and 2 h, respectively, based on the highest bacterial reduction of ∼2.7 log CFU/mL. Following the formation of ST biofilm on cantaloupe at 4 °C and 22 °C, the cantaloupe was treated with phage at an MOI of 100 for 2 h. The antibiofilm efficacy of phage was evaluated via the plate count method, confocal laser scanning microscopy, and scanning electron microscopy (SEM). The initial biofilm population at 22 °C was significantly greater and more condensed than that at 4 °C. After phage treatment, biofilm population and the percentage of viable ST in biofilm were reduced by ∼4.6 log CFU/cm2 and ∼90% within 2 h, respectively, which were significantly greater than those at 22 °C (∼2.0 log CFU/cm2 and ∼45%) (P < 0.05). SEM images also confirmed more drastic destruction of the cohesive biofilm architecture at 4 °C than at 22 °C. As a result of its cold temperature-robust lytic activity and the contribution of endolysin and two depolymerases, vB_SalA_KFSST3 demonstrated excellent antibiofilm efficacy at cold temperature, highlighting its potential as a promising practical biocontrol agent for the control of ST and its biofilm.

Phenotypic and genotypic survey of antibiotic resistance in Salmonella enterica isolates from dairy farms in Uruguay.

Salmonella enterica is an important zoonotic pathogen that is frequently identified in dairy farming systems. An increase in antibiotic resistance has led to inadequate results of treatments, with impacts on animal and human health. Here, the phenotypic and genotypic susceptibility patterns of Salmonella isolates from dairy cattle and dairy farm environments were evaluated and compared. A collection of 75 S. enterica isolates were evaluated, and their phenotypic susceptibility was determined. For genotypic characterization, the whole genomes of the isolates were sequenced, and geno-serotypes, sequence types (STs) and core-genome-sequence types were determined using the EnteroBase pipeline. To characterize antibiotic resistance genes and gene mutations, tools from the Center for Genomic Epidemiology were used. Salmonella Dublin (SDu), S. Typhimurium (STy), S. Anatum (SAn), S. Newport (SNe), S. Agona (Sag), S. Montevideo (SMo) and IIIb 61:i:z53 were included in the collection. A single sequence type was detected per serovar. Phenotypic non-susceptibility to streptomycin and tetracycline was very frequent in the collection, and high non-susceptibility to ciprofloxacin was also observed. Multidrug resistance (MDR) was observed in 42 isolates (56.0%), with SAn and STy presenting higher MDR than the other serovars, showing non-susceptibility to up to 6 groups of antibiotics. Genomic analysis revealed the presence of 21 genes associated with antimicrobial resistance (AMR) in Salmonella isolates. More than 60% of the isolates carried some gene associated with resistance to aminoglycosides and tetracyclines. Only one gene associated with beta-lactam resistance was found, in seven isolates. Two different mutations were identified, parC_T57S and acrB_R717Q, which confer resistance to quinolones and azithromycin, respectively. The accuracy of predicting antimicrobial resistance phenotypes based on AMR genotypes was 83.7%. The genomic approach does not replace the phenotypic assay but offers valuable information for the survey of circulating antimicrobial resistance. This work represents one of the first studies evaluating phenotypic and genotypic AMR in Salmonella from dairy cattle in South America.

Study of Salmonella detection in laying hens using a Bayesian model.

As part of the measures to reduce the prevalence of Salmonella in poultry in the UK, National Control Programmes (NCPs) have been implemented. These involve regular statutory testing of poultry holdings to monitor and estimate the prevalence of Salmonella in the national flock population and to control Salmonella on holdings with positive flocks, especially those serovars most identified with human illness: Salmonella Enteritidis (SE) and S.Typhimurium (ST). It is very important to ensure that the level of testing is appropriate so that it is sufficiently effective to identify positive flocks and to monitor prevalence, but also efficient in the use of resources. The aim of this study was to estimate the sensitivity of both the Operator and Competent Authority (CA) Official sampling used to detect infected flocks, and to also estimate the true proportion of infected holdings of commercial laying flocks in GB each year of the NCP, along with the trend of any changes in prevalence for both SE/ST and non-SE/ST. A Bayesian model was developed to estimate the sensitivity of both Operator and CA Official sampling from the NCP data 2009-2018, and to estimate the true prevalence of infected holdings. The model estimate for the prevalence of infected holdings for the first complete year of the NCP was 3.9% (95% Credible Interval (CI) 2.8-6.2%) for non-SE/ST and 0.8% (95% CI: 0.4%-1.5%) for SE/ST. Prevalence had reduced to 1.6% (non-SE/ST) (95% CI 1.0%-2.5%) and 0.2% (SE/ST) (95% CI 0.1%-0.4%) in 2018. Results indicated a very low sensitivity of Operator sampling (~9%), but a much higher sensitivity of CA Official sampling (~44%). The true prevalence of Salmonella infected holdings in the UK had a mean average reduction of 10.6% (95% CI: 6.3%-15.1%) per annum (non-SE/ST) and 15.9% (95% CI: 6.0%-19.8%) annual reduction for SE/ST. This has shown the effectiveness of the NCP for Salmonella in commercial laying flocks, with reductions in Salmonella overall more or less equal to the target reduction for regulated serovars of 10% per annum. The true prevalence of SE/ST was estimated to be below the final target of less than 2% in every year and was below 0.5% at the end of the 10 year period.

Performance of wild, tailed, humidity-robust phage on a surface-scanning magnetoelastic biosensor for Salmonella Typhimurium detection

A wild, tailed phage (TST) was compared with a genetically modified, filamentous phage (FST) for S. Typhimurium (ST) detection. When both phages were introduced into oppositely charged MUA and MUAM sensors, the RU values of TST showed an obvious increase on the MUAM sensor. The sensitivity of TST [54.78 ΔRU/(log PFU/mL)] was greater than that of FST [48.05 ΔRU/(log PFU/mL)]. The binding affinity (KD = 1.75 × 10−13 M) of TST on MUAM sensor was greater than that of FST. Both phages were specific to only ST, and TST exhibited a persistent binding capability at 50 % RH. When each phage-immobilized sensor was employed on chili pepper, the sensitivity [880.80 Hz/(log CFU/mL)] and detection limit (1.31 ± 0.27 log CFU/mL) of TST were significantly greater than those of FST. The orientation of TST on sensor promoted the uniform capture of bacteria and enhanced the reliable performance of a surface-scanning magnetoelastic biosensor.

Salmonella Typhimurium U32 peptidase, YdcP, promotes bacterial survival by conferring protection against in vitro and in vivo oxidative stress.

YdcP, a U32 peptidase, is characterized as a putative collagenase with a role in several bacterial infections. However, its role in the pathogenesis of Salmonella Typhimurium remains elusive. Here, we investigated the role of U32 peptidase, YdcP, in the intracellular survival of S. Typhimurium (STM). Our study revealed a novel function of YdcP in protecting wild-type Salmonella from in vitro and in vivo oxidative stress. The ydcP knockout strain showed attenuated intracellular proliferation within the murine and human macrophages. Incubation of wild-type Salmonella with H2O2 induced the transcript level expression of ydcP. Moreover, deleting ydcP increased the susceptibility of the bacteria to in vitro oxidative stress. STM ΔydcP showed increased colocalization with the gp91phox subunit of the NADPH phagocytic oxidase in RAW264.7cells. Further, we observed a reduction in the expression of bacterial anti-oxidant genes in STM ΔydcP growing within the RAW264.7cells. The delay in the death of BALB/c mice infected with STM ΔydcP proved the association of ydcP with the in vivo pathogenesis of Salmonella. Finally, the attenuated growth of the ydcP mutant in wild-type C57BL/6 mice and the recovery of their growth inhibition in gp91phox-/- C57BL/6 mice endorsed the role of ydcP in protecting Salmonella from in vivo oxidative stress. Together, our study depicts a novel role of Salmonella Typhimurium YdcP, a putative U32 peptidase in rendering protection against oxidative stress.

Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes.

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella Heidelberg(SH), S. Typhimurium (ST), S. Enteritidis(SE), S. Minnesota(SM), S. Infantis(SI), Salmonella var. monophasic (SVM),APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed β-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.

Polyclonal hen egg yolk antibodies could confer passive protection against Salmonella serotypes in broiler chicks

Non-typhoidal Salmonella serotypes continue to be major food safety and public health threats worldwide. In the era of emerging antimicrobial resistance, it is imperative to search for alternative strategies of infection control. Present study deals with passive protection of young chicks using polyclonal egg yolk antibodies (IgY). Laying hens were actively immunized with heat inactivated Salmonella serotypes Salmonella enterica subsp. enterica serotypes S. Enteritidis (SE) and S. Typhimurium (ST) isolated from the poultry. Serotype specific polyclonal IgY isolated from the hen egg yolk were evaluated for in vitro neutralization potential. Chicks challenged with pathogenic SE and ST were evaluated for passive protection that were co-administered with polyclonal immune IgY. Heat killed antigens (SE/ST) induced protective immune response in the immunized laying hens; and purified immune IgY inhibited the logarithmic phase of Salmonella growth. Administration of egg yolk at 5% (w/w) in the feed containing specific anti-SE or anti-ST polyclonal IgY to Salmonella-challenged chicks resulted in 37.48 and 38.54% reduction in the caecal Salmonella counts. As antimicrobial resistance is emerging as a growing public health problem, findings of this study indicate benefits of prophylactic feeding of egg yolk containing polyclonal IgY for the passive protection of chicks and minimize caecal Salmonella carriage.

Behavior and Immune Response of Conventional and Slow-Growing Broilers to Salmonella Typhimurium.

Fast growth rate in broiler chickens comes with welfare concerns and the contribution of growth rate to pathogen resistance and sickness behavior is relatively unknown. The objective of this study was to evaluate physiological and behavioral responses of conventional (CONV) and slow-growing (SG) male broilers challenged with Salmonella Typhimurium. CONV (n = 156) and SG (n = 156) chicks were raised in a pen with wood litter shavings until day 7 of age, when birds were transferred to 24 isolators (n = 11 chicks/isolator). On day 14 of age, half of the birds (n = 12 isolators) were challenged with S. Typhimurium (ST) and the other half (n = 12 isolators) received a control (C). On days 7, 13, 17, 21, and 24, body weight was recorded, and blood, jejunum and ileum sections were collected from 2 birds/isolator (n = 48 birds/sampling) to measure plasma IgA and IgG and intestinal histomorphology, respectively. On days 12, 16, 21, and 23, video was recorded to evaluate bird postures (sitting, standing, or locomoting) and behaviors (eating, drinking, preening, stretching, sham foraging, allopreening, and aggression). CONV birds were 70 g heavier (p = 0.03) on day 21 and 140 g heavier (p = 0.007) on day 24 than SG. On day 7, CONV jejunum villus height and crypt depth were 22 and 7 μm greater (p ≤ 0.001), respectively, than SG. On day 24, ST ileum villus height was 95 μm shorter (p = 0.009) than C. IgA increased after day 17 for all birds and at day 21, CONV IgA was greater (p = 0.01) than SG. Although SG IgG was 344 μg/ml greater (p = 0.05) than CONV on day 7, CONV IgG increased with age (p < 0.0001) to greater (p ≤ 0.03) concentrations than SG on day 21 and day 24 by 689 μg/ml and 1,474 μg/ml, respectively, while SG IgG remained at similar concentrations after day 13. Generally, a greater proportion of birds sham foraged as they aged (p < 0.0001). A greater proportion of CONV tended to sit (p = 0.09) and fewer locomoted (p < 0.0001) than SG as they aged. The results illustrate conventional and slow-growing broilers differ in their behavior, immunity, and response to Salmonella.

Safety of bivalent live attenuated Salmonella vaccine and its protection against bacterial shedding and tissue invasion in layers challenged with Salmonella

Nontyphoidal Salmonella infection was one of the predominant foodborne illnesses in humans. The medical burden and antimicrobial resistance of salmonellosis gained importance in public health and requested the poultry industry to seek effective measures to control the disease. The objective of this study was to evaluate the safety and effectiveness of a commercial bivalent live attenuated vaccine (AviPro Salmonella DUO) in specific-pathogen-free (SPF) chickens and field layers. It explored its safety and efficacy against medically important strains, Salmonella Enteritidis (SE) and S. Typhimurium (ST). The results demonstrated that ten vaccine doses in SPF chickens and regular doses in commercial layers showed desirable safety without affecting chicken health. Vaccinated layers demonstrated lower flock mortality and higher egg production performance than the unvaccinated layers during the raising and egg production periods. Additionally, no visceral colonization and egg contaminations were detected. Cloacal shedding of vaccine strains was noted, but the colonization of Salmonella disappeared within four weeks of the last vaccination. Regarding vaccine efficacy, one dose significantly reduced Salmonella cloacal shedding (P = 0.037 for SE and P = 0.027 for ST) and viable cell counts (P = 0.003 for SE and ST) on day 7 post the challenges. Significantly low Salmonella loads of cloacal samples on day 14 after the challenges were also determined in the vaccinated group (P = 0.006 for SE; P = 0.041 for ST). Triple immunizations effectively prevented layers from the cloacal shedding on either day 7 or day 14 post Salmonella challenges. Total viable counts of SE and ST in tissues of vaccinated layers were also reduced on day 14 after the challenges (P = 0.026 for SE; P = 0.002 for ST). To conclude, one dose of vaccine exhibited inhibitory effects on Salmonella shedding and tissue invasions in young layers. Following the regimen of triple vaccinations, Salmonella shedding was completely inhibited, and tissue invasions were significantly reduced. Incorporating this vaccine into a comprehensive Salmonella control program is promising to protect layers from the risks of contaminating the flocks and egg products.

Deciphering the role of ttrA and pduA genes for Salmonella enterica serovars in a chicken infection model

ABSTRACT Salmonella enterica serovars use self-induced intestinal inflammation to increase electron acceptor availability and to obtain a growth advantage in the host gut. There is evidence suggesting that the ability of Salmonella to use tetrathionate and 1,2-propanediol provides an advantage in murine infection. Thus, we present here the first study to evaluate both systemic infection and faecal excretion in commercial poultry challenged by Salmonella Enteritidis (SE) and S. Typhimurium (STM) harbouring deletions in ttrA and pduA genes, which are crucial to the metabolism of tetrathionate and 1,2-propanediol, respectively. Mutant strains were excreted at higher rates when compared to the wild-type strains. The highest rates were observed with white egg-layer and brown egg-layer chicks (67.5%), and broiler chicks (56.7%) challenged by SEΔttrAΔpduA, and brown egg-layer chicks (64.8%) challenged by STMΔttrAΔpduA. SEΔttrAΔpduA presented higher bacterial counts in the liver and spleen of the three chicken lineages and caecal contents from the broiler chickens, whereas STMΔttrAΔpduA presented higher counts in the liver and spleen of the broiler and brown-egg chickens for 28 days post-infection (P < 0.05). The ttrA and pduA genes do not appear to be major virulence determinants in faecal excretion or invasiveness for SE and STM in chickens. RESEARCH HIGHLIGHTS ttrA and pudA do not impair gut colonization or systemic infection in chicks. Mutant strains were present in higher numbers in broilers than in laying chicks. Mutants of SE and STM showed greater pathogenicity in broiler chicks than layers.

Impact of Salmonella Control Programmes in Poultry on Human Salmonellosis Burden in Greece

Since 2008, veterinary authorities in Greece have implemented national control programmes (NSCPs) targeting S. Enteritidis (SE) and S. Typhimurium (ST) in poultry. We assessed the effect of the programs on the reported number of human isolates. Using monthly data for 2006–2017, we defined two groups (SE, ST) and one control group with serotypes unrelated to poultry or eggs. For SE we also analysed data for 2006–2015 due to a multi-county SE outbreak in 2016. We performed an interrupted time series analysis and used a negative binominal regression model. For both SE and ST, there was no significant trend of the isolation rate before or after NSCPs’ introduction. After the NSCPs’ introduction there was an increasing rate (IRR: 1.005, 95% CI: 1.001–1.008) for control serotypes and a decreasing one for SE (IRR: 0.990, 95% CI: 0.986–0.995) (for 2009 to 2015 analysis). From 2006 to 2017, NSCPs had a statistically significant impact on the number of SE isolates that decreased by 49% (IRR:0.511, 95% CI: 0.353–0.739). No impact was shown on the number of ST (p-value = 0.741) and control isolates (p = 0.069). As a conclusion, NSCP’s implementation was associated with decreased SE isolates and overall burden of salmonellosis; however further measures aiming at human salmonellosis due to ST, should be considered.

Modulations of growth performance, gut microbiota, and inflammatory cytokines by trehalose on Salmonella Typhimurium-challenged broilers

Salmonellosis in broilers is not merely a significant disease with high economic costs in the poultry industry but also the foodborne disease with the impact on public health by cross-contamination. This study was to investigate the prebiotic ability of trehalose supplementing in diets (0, 1, 3, and 5%, w/w) against Salmonella by using S. Typhimurium (ST)-inoculated broilers. The improvements (P < 0.05) of feed conversion ratio (FCR) were observed with 5% trehalose supplementation in ST-inoculated broilers' diets. An addition of 3 or 5% trehalose in diets increased (P < 0.05) the abundance of lactobacilli in the duodenum and jejunum but decreased (P < 0.05) the growth of ST in the cecum. The adverse effects on serum levels of aspartate aminotransferase, triglyceride, and albumin and globulin ratio in ST-inoculated broilers were noticed and counteracted by supplementing 3 or 5% trehalose in diets (P < 0.05). Besides, the inclusion of trehalose in diets alleviated the intestinal damages and maintained the integrity of cecal epithelial cells after ST challenge under an haematoxylin and eosin-staining observation. Supplementing trehalose further showed the inhibitions of toll-like receptor 4-mediated nuclear factor-kappa-B pathway, including the downregulation (P < 0.05) of proinflammatory cytokine genes, such as interleukin 1 beta and lipopolysaccharide-induced tumor necrosis factor-alpha factor and the upregulation (P < 0.05) of interleukin 10 and interferon-alpha in ST-inoculated broilers. Overall, supplementing trehalose alleviated the adverse effects from ST challenge on FCR, serum biochemistry, the damage, and inflammation in the liver and cecum. Those improvements on ST challenged broilers also contributed to the overgrowth of lactobacilli, the decrement of ST, and anti-inflammatory effects in affected broilers. Trehalose, therefore, could be a promising prebiotic against salmonellosis to benefit broiler production and promote food safety in the poultry industry.

Detection Method for Salmonella Typhimurium and Salmonella Enteritidis using Real-Time Polymerase Chain Reaction

Salmonella is a pathogenic bacterium that can cause serious harm to humans. Chicken carcasses have been reported contaminated by Salmonella, especially S. Typhimurium and S. Enteritidis. These two serovars are very difficult to be confirmed and distinguished using biochemical analysis, therefore a rapid method for detection and differentiation of both is required. The objective of this study was to evaluate designed primer for detection and differentiation of S. Typhimurium and S. Enteritidis on chicken carcasses using real time Polymerase Chain Reaction (rt-PCR). Detection of Salmonella spp. was conducted using primer sequence from invA gene. Differentiation of both Salmonella serovars was conducted using specific target gene from S. Typhimurium (STM) and specific virulence plasmid of S. Enteritidis (Prot6E). The result showed that invA primer effective to detect all species Salmonella tested and has good specificity that could not detect Escherichia coli and Shigella dysenteriae in the similar melting temperature. Two specific primers STM and prot6E have distinguished between S. Typhimurium and S. Enteritidis. Sensitivity of method showed very good with 0.5 μM primer concentration of invA, STM and prot6E that were 0.2 pg, 22 pg and 28 pg respectively. Initial trial showed that this method can be applied for detection of Salmonella spp. and two serovars in chicken carcasses.

Experimental infection of pigs by Salmonella Derby, S. Typhimurium and monophasic variant of S. Typhimurium: Comparison of colonization and serology

Salmonella serovars Derby, Typhimurium and the monophasic variant of Salmonella Typhimurium are the most frequently isolated serovars in pigs in France. To compare the excretion patterns, seroconversion to Salmonella and contamination of the organs of pigs inoculated with strains of all three serovars, we conducted an experimental trial with 28 SPF piglets. Four were used as a negative control, while the other 24 were divided equally into three groups. Each group was inoculated at 7 weeks of age with a different strain: S. Derby (SDb), S. Typhimurium (ST), and the monophasic variant of S. Typhimurium (mST). Fecal and blood samples were collected twice a week up until necropsy, on 21 days post-inoculation (DPI) for half of each group and 49 DPI for the remaining piglets. During necropsy, the tonsils, mesenteric lymph nodes and various intestinal contents were collected from each pig. Salmonella bacteria were quantified in CFU/g by a bacteriological method, and levels of Salmonella antibodies were measured using an ELISA Kit. Piglets inoculated with mST continuously excreted Salmonella in their feces throughout the trial. For each of the other serovars, one piglet was Salmonella-negative on one DPI. The quantity of Salmonella excreted was statistically different between the group inoculated with ST and mST (p < 0.05), but no differences were found between the other serovars. The tonsils, cecum and jejunum were the most contaminated organs in all groups. Seroconversion for all the piglets was completed by different DPI: 28 for ST, 31 for mST and 38 for SDb. No major differences were found in terms of excretion and colonization among the studied serovars.

Salmonella Excludes Salmonella in Poultry: Confirming an Old Paradigm Using Conventional and Barcode-Tagging Approaches.

Salmonella is one of the major foodborne bacterial pathogens, and the consumption of contaminated chicken meats isa primary route of Salmonella transmission into human food chains. However, the mechanism of Salmonella transmission within the chicken flock is not fully understood, including competition among Salmonella strains during chicken infection. The purpose of the present study was to evaluate the competitive exclusion (CE) between different or same Salmonella species consecutively challenged through the oral route. Two different approaches were used to evaluate the CE effect, including tracking Salmonella colonization by wild-type strains with difference in natural antibiotic resistance or DNA barcode-tagged isogenic strains. When day-of-hatch chicks were administered by wild-type S. Typhimurium (ST) on day 1, followed by infection on day 2 by S. Enteritidis (SE) or vice versa, most of the birds were colonized only by the first strains administered (82% by ST or 83% by SE). When similar experiments were performed using two different isogenic barcode-tagged SE strains, Illumina sequencing analysis of the barcode region showed that the first barcode-tagged strains administered were dominant strains, ranging from 92 to 99% of the Salmonella recovered from ceca. These results provide quantitative evidence supporting the CE theory that oral administration of Salmonella will produce predominant inhibition over the subsequent colonization of ceca by the following administration one day later by different or same Salmonella species. We also showed that the use of barcode-tagged isogenic strains in combination with deep profiling of barcodes by Illumina sequencing can serve as a quantitative method for studying complex dynamics of Salmonella infection, transmission and colonization in poultry.

Unsupervised classification of individual foodborne bacteria from a mixture of bacteria cultures within a hyperspectral microscope image

Salmonella is a leading cause of foodborne illness. Traditional detection methods require lengthy incubation periods or expensive reagent kits. Hyperspectral microscope images (HMIs) have been previously investigated as a method for early and rapid detection of bacteria by using a spectral signature that is unique to the organism. Previous HMI use with bacteria has consisted of supervised classification with hypercubes collected for single culture images isolated from highly selective growth media. In order to move forward with HMI as a detection tool in the food industry, unsupervised classification of bacteria cells in mixed culture HMIs was investigated. Four foodborne bacteria cultures, S. Typhimurium (ST) E. coli (Ec), S. aureus (Sa) and L. innocua (Li) were combined in seven different culture combinations with HMIs collected between 450 nm and 800 nm. A k-means divisive cluster analysis (CA) was implemented and mixed culture image sets were found to contain between two and four clusters. CA cluster accuracy was obtained by assigning a dummy variable of the proposed CA classification, then carrying out a discriminant analysis. From the mixed culture HMIs, 700 bacteria cells were classified and accuracies were between 91.92% and 100%, with six of the seven HMI sets resulting in &gt; 97% accuracies. A distance measure between clusters was applied to identify unknown clusters based on single culture reference samples of the four bacteria used. Results showed that the CA has potential for unsupervised classification of bacteria cells, but the distance metric was not an adequate method for identifying the unknown cluster based on reference spectra, potentially due to the collinearity amongst bacteria spectra.

Evaluation of recombinant Salmonella vaccines to provide cross-serovar and cross-serogroup protection

Historically, Salmonella vaccines have been either live attenuated or killed bacterin vaccines that fail to offer cross-serogroup protection, which limits risk mitigation and protection of consumers. Subunit recombinant vaccines which possess highly conserved antigens offer potential to provide cross-serogroup protection, and the ability to express immune-enhancing molecules that promote recognition by the immune system. Six Salmonella subunit vaccine candidates were developed in either attenuated S. Enteritidis (SE) or S. Typhimurium (ST) that cell-surface express antigenic epitopes of high mobility group box 1 immune-enhancing sequence (H), peptidoglycan associated lipoprotein (P), and Omp18 protein Cj0113 (C) in different pattern arrangements for evaluation against S. Heidelberg (SH) challenge in broilers. In exp. 1, chicks were orally vaccinated with SE-CPH, SE-HCP, SE-CHP, ST-CPH, ST-HCP, or ST-CHP at 1×107 cfu/chick, or saline on d 1 and d 14. On d 17 all birds were challenged with 6×106 cfu/chick SH, and ceca collected on d 23 and d 28. On d 23 only SE-CPH reduced (P <0.05) SH recovery at 0.34±0.23 log10 cfu when compared to control at 1.19±0.26 log10 cfu. On d 28, SE-CPH and ST-HCP reduced SH recovery at 0.40±0.40 and 0.51±0.26 log10 cfu, respectively in comparison to control at 1.36±0.23 log10 cfu. For exp. 2, chicks were orally vaccinated with 1×108 cfu/chick SE-CPH, SE-HCP, SE-CHP or saline on d 1. At d 7 all chicks were orally challenged with 7×106 cfu/chick SH and ceca collected on d 28 and d 35. SE-CPH reduced (P <0.05) SH recovery on d 28 when compared to control (6.16±0.13vs. 4.71±0.55 log10 cfu). In exp 3, chicks were vaccinated by spray in a commercial vaccination cabinet with SE-CPH vaccination, 1.6×107 cfu/chick, or saline. Birds were challenged on d 14 with 3×107 cfu/chick SH and ceca collected on d 18 and d 25. SE-CPH reduced SH recovery (P <0.05) on d 18, 2.75±0.05 log10 cfu, and d 25, 1.89±0.43 log10 cfu, as compared to control chickens at 5.6±0.37 (d 18) and 3.98±0.5 log10 cfu (d 25). The results of these experiments suggest that cross-serogroup protection is possible using these SE and ST-vectored subunit vaccines.

Injury and death of various Salmonella serotypes due to acidic conditions

Acid injury could prevent detection of Salmonella in feed and feed-type samples. A previous study showed that after incubation in commonly used pre-enrichment media, mixed feeds and feed ingredients reached a pH (4.0 to 5.0), capable of injuring or killing Salmonella. Approximately 105 colony forming units (CFU) of S. Enteritidis (SE), S. Heidelberg (SH), S. Kentucky (SK), or S. Typhimurium (ST) were individually placed into 50 mL of a citrate buffer at pH of 4, 4.5, or 5.0 for 6 or 24 h at 37°C. After holding at 37°C, each serotype was serially diluted in sterile physiological saline and plated onto xylose lysine tergitol 4 [for injury] and nutrient agar (NA) [for death]. These plates were enumerated and percent injury and/or death determined. The injury and death confirmed that pH 4.0 was detrimental to these Salmonella serotypes. After 24 h at pH 4.0, 60% or more of all 4 serotypes were killed with 100% of S. Kentucky killed. At pH 5.0, 65 to 75% of these organisms were uninjured, with death between 15 to 20%. Researchers testing feed/feed ingredients for Salmonella may not be aware of the acidic nature of the pre-enrichment step and the subsequent injury or death of any Salmonella present, whether healthy or stressed. Current protocols that are considered the “gold standard” may not be detecting Salmonella in samples containing fermentable substrates and extraneous microorganisms, which prevent the accurate detection of Salmonella. Injury or death of a few Salmonella cells could reduce the chance of recovery from any sample. More research is needed with additional Salmonella serotypes and strains to fully understand the challenges of isolating Salmonella from feed.

Salmonella Heidelberg Isolated from Poultry Shows a Novel Resistance Profile

Background: The development of antimicrobial resistance in bacteria is a serious public health issue worldwide. Salmonella spp. is considered a leader cause of gastrointestinal disease in animals and humans, and poultry products have been reported as an important reservoir of the bacterium. S. Heidelberg became lately one of the most prevalent serovars found in several countries. However, hardly any information is available about the epidemiology and the resistance profile of it. Therefore, the objective of this study was to determine the antibiotic resistance profile of S. Heidelberg (SH) and to compare to S. Enteritidis (SE) and S. Typhimurium (ST) isolated from the Southern part of Brazil.Materials, Methods &amp; Results: A total of 162 Salmonella isolates of poultry origin serotyped as SH (54), SE (54), and ST (54) were submitted to the disk-diffusion test with disks containing 10 antibiotics of 7 different classes, routinely used in veterinary and human medicine such as: enrofloxacin, ciprofloxacin, norfloxacin, gentamicin, ceftiofur, ceftriaxone, nalidixic acid, tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol. In addition, the Multi-drug Resistance Pattern (MDRP) and the Multiple-Drug Resistance Index (MDRI) were determined. The Chi-square (χ2) test with 1% of significance level was used to statistically evaluate the results. All isolates were susceptible to norfloxacin, enrofloxacin, and ciprofloxacin. The majority of the isolates were resistant to the quinolone class (68%), more specifically to nalidixic acid, which is considered a synthetic quinolone, followed by penicillin (47%) and cephalosporin (16%). Overall, SH isolates showed higher resistance compared to ST and SE (18, 16.5, and 9.6%, respectively). SH isolated from field samples, mainly drag swabs, showed higher resistance levels (24.2%) than those isolated from slaughterhouses (5.6%). SH showed the highest percentage of resistance to ceftiofur (31.5%), ceftriaxone (9.3%), and tetracycline (64.8%) in comparison to the other two serotypes. Most of the SH isolates were resistant to at least two (66.7%), three or more antibiotics (33.3%). A different scenario was observed for ST and SE, where 25.9 and 9.3% were susceptible to at least one drug, respectively. The most common pattern of resistance (MDRP) was C (gentamicin - nalidixic acid - tetracycline) for 14 SH isolates, and A (ceftiofur - nalidixic acid - tetracycline) for 12 SH isolates. MDRI indicated that 22.8% of all isolates were multidrug resistant. SH was the isolate with the largest variety of resistance patterns compared to ST and SE, where 11.7% of the isolates were resistant to more than three antibiotics. In addition, SH showed the greatest MDRI (0.25) ranging from 0.2 to 0.5.Discussion: SH was resistant to almost all antibiotics tested and showed multi-drug resistant profile, therefores, it showed a potential for horizontal transmission of resistance genes. Additionally, SH showed a higher resistance profile for ceftiofur, an important antibiotic used in poultry, which can cross-resist to ceftriaxone, commonly used to treat salmonellosis in children. Our results showed that SH is a real challenge regarding antimicrobial resistance. This scenario leads to the need for rational and judicious use of antimicrobials in poultry and, as an alert to the medical community.